PROJECT SUMMARY Many rod-shaped bacteria are polarized, meaning that one end of the cell is morphologically and functionally distinct from the other, but there is a general lack of understanding of the mechanisms by which polar asymmetry is established and maintained. Since polarized features provide critical spatial cues for many processes, including chromosome segregation and cell growth, learning the molecular basis for this `bacterial anatomy' will provide fundamental advancements in prokaryotic cell physiology. Furthermore, some pathogenic bacteria use cell polarity to create multiple cell types with specialized roles in supporting virulence. Understanding cell polarity in this context may therefore provide fundamental discoveries relating to core mechanisms for bacterial pathogenicity. What keeps the poles in a state of disequilibrium? This project focuses on the molecular mechanisms that control the interactions between polar proteins and a polar organizing protein, called PopZ, which forms polymeric scaffolds at both cell poles in Caulobacter crescentus. The approach extends from the surprising discovery that PopZ is a molecular hub that organizes the cell by directly interacting with a large number of binding partners. This implies that there is a selection mechanism that supports two parallel programs for PopZ-dependent multiprotein complex assembly at opposite ends of the cell. The proposal includes a series of experiments that will elucidate the selection mechanism. Using a group of established PopZ binding partners, structural and biophysical analyses will be used to understand binding and binding kinetics at the atomic scale. An area of particular focus will be an intrinsically disordered region within PopZ that determines binding specificity. These experiments will provide critical information in developing a detailed biophysical model of hub network assembly and function. The second aim is to test different models for establishing and maintaining polarity in Caulobacter. One possibility is that stable binding partners are used to mark an `old' pole, another is that each pole has a different set of signaling proteins that reinforce polarity through signal feedback loops.